SummaryIn response to stress, cancer cells generate nutrients and energy through a cellular recycling process called autophagy, which can promote survival and tumor progression. Accordingly, autophagy inhibition has emerged as a potential cancer treatment strategy. Inhibitors targeting ULK1, an essential and early autophagy regulator, have provided proof of concept for targeting this kinase to inhibit autophagy; however, these are limited individually in their potency, selectivity, or cellular activity. In this study, we report two small molecule ULK1 inhibitors, ULK-100 and ULK-101, and establish superior potency and selectivity over a noteworthy published inhibitor. Moreover, we show that ULK-101 suppresses autophagy induction and autophagic flux in response to different stimuli. Finally, we use ULK-101 to demonstrate that ULK1 inhibition sensitizes KRAS mutant lung cancer cells to nutrient stress. ULK-101 represents a powerful molecular tool to study the role of autophagy in cancer cells and to evaluate the therapeutic potential of autophagy inhibition.

The search for a single silver bullet for the treatment of cancer has now been overshadowed by the identification of multiple therapeutic targets unique to each malignancy and even to each patient. In recent years, autophagy has emerged as one such therapeutic target. In response to both therapeutic and oncogenic stress, cancer cells upregulate and demonstrate an increased dependence upon this intracellular recycling process. Particularly in malignancies that currently lack targeted therapeutic options, autophagy inhibitors are the next hopeful prospects for the treatment of this disease. In this review, we discuss the rapid evolution of autophagy inhibitors from early lysosomotropic agents to next-generation lysosome-targeted drugs and beyond.

Summary In cancer, autophagy is upregulated to promote cell survival and tumor growth during times of nutrient stress and can confer resistance to drug treatments. Several major signaling networks control autophagy induction, including the p53 tumor suppressor pathway. In response to DNA damage and other cellular stresses, p53 is stabilized and activated, while HDM2 binds to and ubiquitinates p53 for proteasome degradation. Thus blocking the HDM2-p53 interaction is a promising therapeutic strategy in cancer; however, the potential survival advantage conferred by autophagy induction may limit therapeutic efficacy. In this study, we leveraged an HDM2 inhibitor to identify kinases required for p53-dependent autophagy. Interestingly, we discovered that p53-dependent autophagy requires several kinases, including the myotonic dystrophy protein kinase-like alpha (MRCKα). MRCKα is a CDC42 effector reported to activate actin-myosin cytoskeletal reorganization. Overall, this study provides evidence linking MRCKα to autophagy and reveals additional insights into the role of kinases in p53-dependent autophagy.

Hydroxychloroquine (HCQ) is a 4-aminoquinoline molecule used for the treatment of malaria, and more recently to treat rheumatoid arthritis, systemic lupus erythematosus, and cancer. In cancer, HCQ is being used in multiple cancer clinical trials as an inhibitor of autophagy, a cytosolic degradation process employing the lysosome. Importantly, more potent lysosomotropic agents are being developed as autophagy inhibitors. Additional studies revealed that acridine-based compounds such as quinacrine (QN) increased potency over the 4-aminoquinoline HCQ. In line with these initial discoveries, we performed chemical synthesis of acridine-based compounds and screened for potent autophagy inhibition. The novel compound VATG-027 increased potency and cytotoxicity over HCQ in osteosarcoma and melanoma cell lines, supporting further investigation in vivo. Here, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to investigate HCQ, QN, and VATG-027 compound concentrations across various tissue types in mice. This method detected compound concentrations in whole blood, lung, liver, kidney, and subcutaneous tumor tissues. Concentrations of HCQ, QN, and VATG-027 varied within and between tissue types, suggesting unique tissue distribution profiles for 4-aminoquinoline and acridine compounds.